Artificial antigen-presenting cells and methods for producing and using the same

ABSTRACT

Described herein are biomimetic Janus particles useful as artificial antigen presenting cells capable of activating T cells in vitro. “Bull&#39;s eye” ligand patterns mimicking either the native or reverse organization of the T cell immunological synapse are provided on the surface of nano- or micro-sized particles. Methods for activating T cells in vitro using biomimetic Janus particles described herein are also provided. T cells activated by the biomimetic Janus particles can be used in adoptive immunotherapies for treating cancer, tolerance induction in autoimmune disease, autologous immune enhancement therapy, and viral infection immunotherapy. Also described herein are methods for producing a biomimetic Janus particle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/513,143, filed Mar. 21, 2017, which is a U.S. National StageApplication of International Patent Application No. PCT/US2015/051756,filed Sep. 23, 2015, which claims the benefit of U.S. ProvisionalApplication No. 62/054,832, filed Sep. 24, 2014, each of which isexpressly incorporated herein by reference in its entirety.

BACKGROUND

T cells, as the key player in cell-mediated immunity, can be harnessedto fight against cancer, induce tolerance in autoimmune disease, enhanceimmune therapy, and to fight against viral infection. An essential stepin employing T cells for use these therapies, is to stimulate T cellactivation in vitro. T cells in vivo are stimulated byantigen-presenting cells (APCs), such as dendritic cells. However,isolation of native APCs for T cell-based immunotherapy is not onlytime-consuming and expensive, but also difficult to reproduce. Toovercome those challenges, synthetic particles that display well-definedantigens and co-stimulatory ligands have been developed as artificialAPCs to provide more consistent stimulation of T cells in vitro. A largespectrum of particles has been designed to mimic different aspects ofthe native APCs, including size, shape, ligand mobility, andmultivalency. However, these particles are generally randomly coatedwith proteins, and do not truly mimic the native immunological synapse.

SUMMARY

Described herein are biomimetic Janus particles useful as artificialantigen presenting cells capable of activating T cells in vitro. “Bull'seye” ligand patterns mimicking either the native or reverse organizationof the T cell immunological synapse are provided on the surface of nano-or micro-sized particles. Methods for activating T cells in vitro usingbiomimetic Janus particles described herein are also provided. T cellsactivated by the biomimetic Janus particles can be used in adoptiveimmunotherapies for treating cancer, tolerance induction in autoimmunedisease, autologous immune enhancement therapy, and viral infectionimmunotherapy. Also described herein are methods for producing abiomimetic Janus particle.

In a particular embodiment provided herein is a biomimetic Janusparticle comprising a particle, at least one substantially concentricpattern of a first ligand population bound to the particle, wherein thefirst ligand population comprises one or more ligands involved in T cellactivation and/or one or more molecules to which a ligand involved in Tcell activation may be bound, and a second ligand population bound tothe particle, wherein the second ligand population is different than thefirst ligand population and does not substantially overlap thesubstantially concentric pattern of the first ligand population, andwherein the second ligand population comprises one or more ligandsinvolved in T cell activation and/or one or more molecules to which aligand involved in T cell activation may be bound, wherein one of thefirst ligand population or second ligand population comprise anintegrin-binding ligand.

In another particular embodiment provided herein is a method forproducing a biomimetic Janus particle, the method comprising the stepsof a) forming at least one substantially concentric pattern of a firstligand population on at least one particle, wherein the first ligandpopulation comprises one or more ligands involved in T cell activationand/or one or more molecules to which a ligand involved in T cellactivation may be bound, and b) incubating the at least one particlefollowing completion of step a) with a second ligand population, whereinthe second ligand population is different than the first ligandpopulation and does not substantially overlap the substantiallyconcentric pattern of the first ligand population, and wherein thesecond ligand population comprises one or more ligands involved in Tcell activation and/or one or more molecules to which a ligand involvedin T cell activation may be bound. The method for forming the at leastone substantially concentric pattern of the first ligand population onthe at least one particle can be any form of nano- or microlithography,including but not limited to microcontact printing, photolithography,electron beam lithography, nanoimprint lithography, interferencelithography, X-ray lithography, extreme ultraviolet lithography,magnetolithography, and dip-pen lithography.

In embodiments using microcontact printing to form the at least onesubstantially concentric pattern of the first ligand population on theat least one particle, the method comprises the steps of a) forming amonolayer of particles on a substrate, b) providing a section of curedpolymer, c) incubating a surface of the section of cured polymer with afirst ligand population, wherein the first ligand population comprisesone or more ligands involved in T cell activation and/or one or moremolecules to which a ligand involved in T cell activation may be bound.d) drying the cured section of polymer incubated with the first ligandpopulation, e) embedding the monolayer of particles in the dried, curedsection of polymer incubated with the first ligand population bypressing the dried, cured section of polymer incubated with the firstligand population against the monolayer of particles, f) removing theparticles from the substrate, wherein the particles either remainembedded in, or are released from, the dried, cured section of polymerincubated with the first ligand, and g) incubating the particles with asecond ligand population, wherein the second ligand population comprisesone or more ligands involved in T cell activation and/or one or moremolecules to which a ligand involved in T cell activation may be bound.

In embodiments wherein the particles remain embedded in the dried, curedsection of polymer incubated with the first ligand population, themethod further comprises a step of releasing the particles from thedried, cured section of polymer incubated with the first ligandpopulation following the incubation with the second ligand population,so as to reveal a substantially concentric pattern of the first ligandpopulation formed on the particle substantially surrounded by the secondligand population. In certain embodiments, the released particles arere-incubated with the second ligand population.

In embodiments wherein the particles are released from the dried, curedsection of polymer incubated with the first ligand population, whereinsteps a) through f) are repeated with the particles one or more timesbefore completing step g), so as to produce two or more substantiallyconcentric patterns of the first ligand population formed on theparticle substantially surrounded by the second ligand population.

The substrate used for formation of the monolayer can be any substratesuitable for particle monolayer formation. In one embodiment, thesubstrate is glass, and more specifically, a glass microscope slide. Incertain embodiments, the substrate is cleaned prior to the formation ofthe monolayer of particles thereon. The substrate can be cleaned with asolvent to remove organic residues and to hydroxylate the surface of thesubstrate, making it hydrophilic. In one embodiment, the solvent used toclean the substrate is piranha solution comprising H₂SO₄/H₂O₂. Inanother embodiment, the substrate is cleaned at an elevated temperatureof 5° C. for about 15 minutes. Following cleaning, the substrate isgenerally rinsed. In one embodiment, the substrate is rinsed inultrapure water following cleaning.

In certain aspects, the particles are cleaned prior to forming themonolayer of particles on the substrate. In one aspect, the particlesare cleaned using piranha solution comprising H₂SO₄/H₂O₂. Formation ofthe monolayer of particles on the substrate is, in certain embodiments,accomplished via solvent evaporation or dip coating.

In certain embodiments described herein, the cured polymer is asilicon-based organic polymer, such as polydimethylsiloxane. The curedpolymer has an area sufficient to contact the microlayer of particles onthe substrate, and is generally about 0.5 cm² to about 10 cm². Incertain aspects, the surface of the cured polymer is treated to make itssurface hydrophilic. This provides for improved adsorption of the firstligand population to the cured polymer. In certain aspects, this isachieved by treating the cured polymer with H₂SO₄/H₂O₂. The dried, curedsection of polymer incubated with the first ligand population is pressedagainst the monolayer of particles immediately following drying. Incertain aspects, the dried, cured section of polymer incubated with thefirst ligand population is pressed against the monolayer of particles ata pressure of about 0.5×10⁴ to about 2.5×10⁴ Pa. In a particularembodiment, the dried, cured section of polymer incubated with the firstligand population is pressed against the monolayer of particles at apressure of about 1.5×10⁴ Pa. The dried, cured section of polymerincubated with the first ligand population is pressed against themonolayer of particles for about 30 seconds to about 10 minutes. Thediameter of first ligand population deposited on the particle isdirectly related to the pressure applied to the cured section of polymerincubated with the first ligand population when pressed against themonolayer of particles.

In other embodiments, the particles embedded in the dried, cured sectionof polymer incubated with the first ligand population are incubated inthe second ligand population for about 0.5 to about 3 hours. In aparticular embodiment, the particles embedded in the dried, curedsection of polymer incubated with the first ligand population areincubated in the second ligand population for about 1.5 hours.

In another particular embodiment provided herein is a method forproducing a biomimetic Janus particle using block copolymerself-assembly. Generally, the method comprises the steps of a) providinga first block copolymer to which is bound a first ligand population,wherein the first ligand population comprises one or more ligandsinvolved in T cell activation and/or one or more molecules to which aligand involved in T cell activation may be bound, b) providing a secondblock copolymer, and c) combining the first block copolymer to which isbound the first ligand population with the second block copolymer,wherein the first block copolymer to which is bound the first ligand andthe second block copolymer self-assemble to form at least onesubstantially spherical biomimetic Janus particle having at least onesubstantially concentric pattern of the first block copolymer to whichis bound the first ligand population. In certain aspects, the bockcopolymers undergo directed self-assembly.

In certain embodiments, a second ligand population is bound to thesecond block copolymer prior to completing the combining step. In otherembodiments, the at least one substantially spherical biomimetic Janusparticle is incubated with a second ligand population.

Particles described herein can be microparticles or nanoparticles. Incertain embodiments, the particle is selected from the group consistingof: a silica particle; a polystyrene particle; a melamine resinparticle; and a polymethacrylate particle. In a particular aspect, theparticle is a silica particle. The particles have a diameter within therange of about 0.1 μm to about 20 μm. In a particular aspect, theparticles have a diameter of about 3 μm.

The at least one substantially concentric pattern of the first ligandpopulation has a diameter within the range of about 10 nm to about 5 μm.In one particular aspect, the at least one substantially concentricpattern of the first ligand population has a diameter within the rangeof about 1.7 μm.

In certain embodiments described herein, the one or more ligandsinvolved in T cell activation is selected from the group consisting of:anti-CD3 antibody; anti-CD28 antibody; anti-TCR antibody; anti-CTLA4antibody; and a ligand comprising a general integrin-binding motif, andthe one or more molecules to which a ligand involved in T cellactivation may be bound is biotin. In a particular aspect, the firstligand population comprises one or more ligands comprising a generalintegrin-binding motif and the second ligand population comprises one ormore ligands capable of binding to at least one component of a T cellTCR complex, forming a reverse bull's eye pattern. In another particularaspect, the first ligand population comprises one or more ligandscapable of binding to at least one component of a T cell TCR complex andthe second ligand population comprises one or more ligands comprising ageneral integrin-binding motif, forming a native bull's eye pattern. Theligand comprising a general integrin-binding motif is selected from thegroup consisting of: fibronectin; collagen; laminin; vitronectin;fibrinogen; and thrombospondin.

In embodiments comprising biotin as one of the ligands, the biomimeticJanus particle or method further comprises one or morestreptavidin-conjugated ligands and/or biotinylated ligands capable ofbinding to at least one component of a T cell TCR complex, wherein theone or more streptavidin-conjugated ligands is bound to the biotin.

In certain embodiments, the one or more ligands capable of binding to atleast one component of a T cell TCR complex is selected from the groupconsisting of: anti-CD3 antibody; anti-CD28 antibody; anti-TCR antibody;and anti-CTLA4 antibody.

In other particular embodiments provided herein, are biomimetic Janusparticles produced by a method described herein. In yet otherembodiments provided herein are compounds comprising a biomimetic Janusparticle described herein.

In another particular aspect provided herein, is a method of activatinga population of T cells in vitro, comprising applying one or morebiomimetic Janus particles described herein to a population of T cells.The population of T cells can be autologous T cells, isolated from asubject in need of an immunotherapy, heterologous T cells derived from asource other than the subject in need of an immunotherapy, or acombination thereof. The population of T cells can comprise naïve Tcell, CD8⁺ T cells, CD4⁺ T cells, or a combination thereof. In otherembodiments, the population of T cells comprises antigen-specific Tcells. In yet other embodiments, the population of T cells comprisestumor antigen-specific T cells.

In certain embodiments, the one or more biomimetic Janus particles areincubated with the population of T cells for about 5 minutes to about 2weeks. In a particular aspect, the one or more biomimetic Janusparticles are incubated with the population of T cells for about 1 hourto about 60 hours. In another particular aspect, the one or morebiomimetic Janus particles are incubated with the population of T cellsfor about 24 hours to about 48 hours.

In yet another particular aspect provided herein, is a method ofadministering an immunotherapy to a subject in need thereof, the methodcomprising administering T cells activated by a biomimetic Janusparticle described herein to the subject. The immunotherapy is selectedfrom the group consisting of: adoptive immunotherapy for cancer;tolerance induction in autoimmune disease; autologous immune enhancementtherapy; and viral infection immunotherapy. In certain embodiments, themethod of administering an immunotherapy to a subject in need thereoffurther comprises a step of preparative lymphodepletion in the subjectprior to administering the activated T cells to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: “Bull's eye” Janus particles resemble protein patterns inthe immunological synapse. FIG. 1A: Schematic representation ofligand-bound T cell receptors (TCRs) and integrins forming “bull's eye”concentric microdomains in the membrane junction, known as theimmunological synapse, between a T cell and an antigen-presenting cell.FIG. 1B: Schematic representation of patterns of anti-CD3 andfibronectin on Janus particles resembling the native or reverse “bull'seye” pattern.

FIG. 1C: “Bull's eye” Janus particles resemble protein patterns in theimmunological synapse. Schematic representation of the microcontactprinting of “bull's eye” patterns on particles.

FIGS. 1D-1E: “Bull's eye” Janus particles resemble protein patterns inthe immunological synapse. 3-D confocal fluorescence images of the“bull's eye” Janus particles.

FIG. 1F: Schematic representation of “bull's eye” Janus particlespossessing either one, two, or multiple substantially concentricpatterns of a first ligand (red) surrounded by a second ligand (green).

FIGS. 2A-2B: 3-D fluorescence confocal images show the intracellularclustering of actin (FIG. 2A) and protein kinase C (PKC)-θ (FIG. 2B) inT cells that were stimulated by the “bull's eye” Janus particlesdescribed herein. Clustering morphologies of actin and PKC-θ aresummarized into three categories: focal, diffusive, and annular, asindicated by the schematic symbols in the bar graphs on the right ofeach figure. The blue color in each symbol indicates the localization ofeither actin or PKC-θ with respect to the “bull's eye” pattern on theparticles. Scale bars: 5 μm.

FIGS. 3A-3D: Data showing intracellular calcium elevation in T cellsthat were stimulated by the reverse (FIGS. 3A-3B) and the native (FIGS.3C-3D) “bull's eye” Janus particles. Jurkat T cells were loaded withcalcium-sensitive dye, Fluo-4 AM, whose fluorescence intensity increaseswith intracellular concentration of Ca²⁺. FIGS. 3A and 3C: Normalizedfluorescence intensities of two representative cells are plotted as afunction of time to show the fluctuation of [Ca²⁺] during T cellactivation. Time zero was defined as the time of each cell landing onthe bottom of imaging chambers. White arrows indicate Janus particlesthat were in contact with cells. Anti-CD3 and fibronectin were shown inred and green, respectively, in both images. The global calcium responseof T cells stimulated by (FIG. 3B) the reverse (n=96) and (FIG. 3D) thenative “bull's eye” Janus particles (n=111) are plotted on a color scaleand sorted based upon the fluorescence intensity of the first peak.Scale bars: 5 μm.

FIGS. 4A-4B: Bar graphs showing size distribution of anti-CD3 (FIG. 4A)and fibronectin (FIG. 4B) patches.

FIG. 5: 3-D fluorescence confocal images show intracellular clusteringof actin in T cells stimulated by control particles that were uniformlycoated with anti-CD3 and fibronectin. The images are representative of36 cells. Scale bars: 5 μm.

FIG. 6: 3-D fluorescence confocal images show intracellular clusteringof PKC-θ in T cells stimulated by control particles that were uniformlycoated with anti-CD3 and fibronectin. Scale bars: 5 μm.

FIG. 7: Data showing intracellular calcium elevation in Jurkat T cellsthat were stimulated by particles coated with anti-CD3 (top panel),fibronectin (middle panel), and BSA (bottom panel). Ligandfunctionalization was uniform for all three control samples. Jurkat Tcells were loaded with calcium-sensitive dye, Fluo-4, whose fluorescenceintensity increases with intracellular Ca²⁺ concentration. Normalizedfluorescence intensities of individual cells are shown on a color scale.Time zero is defined as the time of each cell landing on the bottom ofimaging chambers.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced. The disclosures of thesepublications, patents and published patent specifications are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

Described herein are biomimetic Janus particles useful as artificialantigen presenting cells capable of activating T cells in vitro. “Bull'seye” ligand patterns mimicking either the native or reverse organizationof the T cell immunological synapse are provided on the surface of nano-or micro-sized particles. Methods for activating T cells in vitro usingbiomimetic Janus particles described herein are also provided. T cellsactivated by the biomimetic Janus particles can be used in adoptiveimmunotherapies for treating cancer, tolerance induction in autoimmunedisease, autologous immune enhancement therapy, and viral infectionimmunotherapy. Also described herein are methods for producing abiomimetic Janus particle.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, the term “biomimetic” refers to artificially mimicking anatural molecule, compound, or particle. A biomimetic particle describedherein has substantially similar T cell activating abilities as a nativeantigen presenting cell. In a particular embodiment, a biomimeticparticle comprises first and second ligand populations. The ligandpopulations comprise one or more ligands involved in T cell activation,one or more molecules to which a ligand involved in T cell activationmay be bound, or a combination thereof. Generally, the first ligandpopulation will be different than the second ligand population. Incertain aspects both the first and second ligand populations cancomprise of one or more of the same ligands, but will differ in at leastone ligand.

The term “ligand” generally refers to a substance, including a protein,that forms a complex that binds specifically and reversibly to anotherchemical entity to form a larger complex. As used herein, a ligand isusually a signal-triggering molecule, binding to a site on a targetprotein. As described herein, ligands include any antibody or otherligand capable of binding to a T cell target protein involved in T cellactivation or signaling. Ligands may include, but are not limited to,anti-CD3 antibodies, anti-CD28 antibodies, anti-T cell receptorantibodies, ligands comprising a general integrin-binding motif, andbiotin. Ligands comprising a general integrin-binding motif generallycomprise an arginine-glycine-aspartic acid (RGD) motif, although maycomprise a different integrin-binding motif. Ligands comprising ageneral integrin-binding motif include, but are not limited to,fibronectin, collagen, laminin, vitronectin, fibrinogen, andthrombospondin. “Ligand population” refers to a group of ligands. Aligand population can consist of a single type of ligand, or maycomprise several types of ligands. For example, a ligand population maycomprise one or more unique anti-CD3 antibodies, each anti-CD3 antibodyin the ligand population capable of interacting with one or more CD3subunits.

As used herein “Janus particle” refers to a particle whose surface hastwo or more distinct physical properties. A Janus particle may comprisea biomimetic particle, wherein the Janus particle comprises twodistinct, bound ligand populations, thus possessing the properties andfunctionalities of the two bound ligand populations. The two ligandpopulations are generally arranged in a “bull's eye” configuration on aJanus particle, giving the particle biomimetic properties substantiallysimilar to those of a natural antigen presenting cell. The term “bull'seye” configuration refers to a patterning of ligand populations whereone or more substantially concentric patterns of the first ligandpopulation being surrounded by the second ligand population. In certainaspects, the second ligand population does not significantly overlapwith the concentric pattern formed by the first ligand population.

The term “modulating T cell activation,” as used herein, refers tomanipulating T cell signaling pathways, thereby affecting T cellactivation. Preferably, the term refers to initiating signaling in Tcells through the T cell activation pathway, involving simultaneousengagement of a T cell receptor and a costimulatory receptor. Examplesof T cell costimulatory receptors include, but are not limited to, CD28,CTLA4, Inducible Costimulator (ICOS), and integrins, including but notlimited to, VLA-4, VLA-5, and LFA-1. Modulation of T cell activationoccurs at the T cell's T cell receptor (TCR) complex. A T cell TCRcomplex generally comprises the TCR, and CD3, and may comprise one ormore costimulatory receptors.

“Particles,” as used herein, generally refers to synthetic nano- ormicro-sized particles of about 0.1 μm to about 100 μm in diameter.Particles used herein may be any size of about 0.1 μm and about 100 μmin diameter. In certain embodiments, particles are about 0.1 μm to about20 μm in diameter. In other embodiments, particles are about 0.5 μm toabout 5 μm in diameter. In a particular embodiment, particles are about3 μm in diameter. The term “particles” may refer to a synthetic particlecomprised of various materials, including but not limited to, silica,polystyrene, melamine resin, and polymethacrylate.

As used herein, the term “monolayer” when used to describe anarrangement of particles, refers to a single, closely packed layer ofthe particles.

As used herein, the term “substantially concentric” means a shape beingapproximately circular. A substantially concentric shape need not beperfectly circular, but may vary in radius as measured from pointsaround the circumference of the shape. The term “substantiallyconcentric” includes, but is not limited to, ellipses and ovoids.Further, a “substantially concentric” shape need not have a smoothcircumference.

As used herein, “ultrapure water” refers to water that has been treatedto the highest levels of purity for all types of contaminants including,but not limited to organic and inorganic compounds, dissolved andparticulate matter, and dissolved gases.

“Solvent evaporation” refers to the process of applying a solventcontaining a substance, such as microparticles, to a substrate andevaporating the solvent. Solvent evaporation can be employed to form amonolayer of microparticles on a substrate.

“Dip coating” refers to the process of introducing a substrate to acolloidal suspension comprising particles, such as microparticles, andwithdrawing the substrate, thereby forming a uniform liquid filmcontaining microparticles. Following evaporation of a solvent, a uniformmonolayer of microparticles remains on the substrate.

As used herein, the term “about” refers to an amount varying from thestated value or range by 1/10 of the stated value or range, but is notintended to limit any value or range of values to only this broaderdefinition. For example, a microparticle having a diameter of about 3 μmincludes microparticles having diameters of 2.7 to 3.3 μm. Each value orrange of values preceded by the term “about” is also intended toencompass the embodiment of the stated absolute value or range ofvalues.

As used herein, the term “immunotherapy” refers to a therapy thatinduces, enhances, or suppresses the immune response in a subject. Inparticular embodiments, an immunotherapy may include, but is not limitedto, adoptive immunotherapy, tolerance induction in an immune disease,autologous immune enhancement, and viral infection immunotherapy. Inparticular embodiments, an immunotherapy is administered to a subjectenterally, parentally, intramuscularly, intravesically, subcutaneously,or transmucosally.

As used herein, “adoptive immunotherapy” refers to an immunotherapywherein immune-derived cells are transferred into a subject with thegoal of transferring the immunologic functionality and characteristicsof the transferred immune-derived cells into the subject. Adoptiveimmunotherapy may be harnessed to treat a number of conditions,including but are not limited to, cancer, autoimmune disease, and viralinfection, as well as to induce tolerance in autoimmune disease.

As used herein, “tolerance induction” refers to the initiation orcausing of refractivity to activating receptor-mediated stimulation.Such refractivity is generally antigen-specific and persists afterexposure to a tolerizing antigen has ceased. Tolerance to self-antigensor to foreign antigens can be induced. In one particular embodiment, atolerant subject does not produce an adverse immune response to aself-antigen over a period of time after treatment with a T cellactivated by a biomimetic particle described herein ceases, even whenchallenged with the antigen, and remains capable of providing an immuneresponse against other antigens.

As used herein, “autologous immune enhancement therapy” refers to aparticular adoptive immunotherapy wherein immune-derived cells are takenfrom a subject (autologous cells), cultured, and processed to activatethe cells, and then reintroduced into the subject. In particularembodiments, the autologous cells are activated by a biomimetic particledescribed herein.

As used herein, “viral infection immunotherapy” refers to the use ofimmune derived cells as a therapeutic to initiate or supplement animmune response in a subject suffering from a viral infection. Inparticular embodiments, the immune-derived cells used in a viralinfection immunotherapy are heterologous antigen-specific cells.Preferably, the immune-derived cells used in a viral infectionimmunotherapy are autologous antigen-specific cells. Immune derivedcells used in a viral infection immunotherapy are preferably activatedby a biomimetic particle described herein.

“Preparative lymphodepletion” refers to the temporary ablation of theimmune system of a subject in preparation for an immunotherapy (e.g.,adoptive cancer immunotherapy). Total-body irradiation or chemotherapycan be used to deplete lymphoid cells. Preparative lymphodepletion isknown to augment the efficacy of tumor-specific T cells in thelymphopenic environment. Preparative lymphodepletion has been furthershown to enhance the effectiveness of adoptive immunotherapy bydepleting endogenous cells that compete for activating cytokines, and bydepleting endogenous regulatory T cells which diminish the effectivenessof the therapy.

“Autologous T cells” are T cells derived from the same subject to whichthe T cells will be reintroduced.

“Heterologous T cells” are T cells derived from a source other that thesubject to which the T cell will be introduced.

General Description

T cells are activated when T cell receptors (TCRs) and the co-receptorsbind their respective ligands on the surface of antigen presenting cells(APCs). The initial ligand-receptor recognition leads to the formationof a micron-sized membrane junction known as the immunological synapse.As T cell signaling proceeds, ligand-bound membrane receptors andsignaling proteins, such as TCRs, co-stimulatory receptors and adhesionmolecules, are clustered and reorganized in the synapse to form apattern of several distinct concentric domains (FIG. 1A). Ligand-boundTCRs are transported from the cell periphery to accumulate in thecentral domain of the immunological synapse, while integrins and manycytoskeletal proteins are enriched in a ring structure surrounding theTCRs. The protein spatial organization is a reflection of long-rangemolecular interactions that take place in T cell activation.Additionally, the immunological synapse formation, by controlling whichproteins come together and when they are apart, is a regulatorycheckpoint for T cell stimulation.

Synthetic particles that display well-defined antigens andco-stimulatory ligands have been developed as artificial APCs (aAPCs) tostimulate T cell activation in vitro. These aAPCs have been designed tomimic different aspects of the native APCs, including size shape, ligandmobility, and multivalency. However, current design of these aAPCs hasmainly focused on particles that have uniformly distributed ligands.Yet, T cell activation is known to involve spatial segregation ofproteins. And while two-dimensional studies have demonstrated apotential for modulating T cell activation by spatially manipulating theimmunological synapse formation, they cannot mimic the three-dimensionalstructure of native APCs.

The data disclosed herein demonstrates the ability of ligands patternedon nano- or micro-sized particles to mimic either the native or reverseimmunological synapse (FIG. 1B), resulting in effective artificial APCs(aAPCs). The biomimetic aAPCs described herein, or biomimetic Janusparticles, are useful in the activation of T cells in vitro. Theactivated T cells are useful in various T cell-based immunotherapies.

Named after the two-faced Roman god, Janus particles, by possessingdistinct surface makeups or compartments in one entity, offer manypromising biomedical applications that are not possible with homogeneousparticles. For example, multicompartmental Janus particles have beendeveloped as drug delivery carriers that allow controlled step-wise drugrelease. Multiplexed biomolecular detection is possible when one side ofa Janus particle captures analytes while the other side is graphicallyencoded. Janus particles that are half-magnetic and half-fluorescentenable simultaneous imaging and magnetic therapy. Bi-functionality ofJanus particles can also be exploited for simultaneous imaging/sensing.A new application of Janus particles as aAPCs for immune cell activationis described herein.

Bull's eye protein patterns that mimic either the native or reverseimmunological synapse on the surface of micron-sized Janus particles aredescribed herein (FIG. 1B). The two types of patterned particlesactivate T cells by directing the spatial organization of signalingproteins. The native and reverse bull's eye protein patterns describedherein are shown herein to direct intracellular clustering of signalingproteins, such as actin and protein kinase C (PKC)-θ. T cell signaling,as indicated by intracellular calcium ion elevations, also differeddepending on the ligand pattern. The data presented herein demonstratesthe ability to use micropatterned Janus particles as aAPCs to modulate Tcell signaling and activation.

In certain embodiments, a biomimetic Janus particle comprises asubstantially concentric deposition of a first ligand populationsurrounded by a second ligand population. In other embodiments, thebiomimetic Janus particle comprises two or more substantially concentricdepositions of the first ligand population. When two or moresubstantially concentric depositions are of the first ligand populationare present, they are interspersed, or “filled in,” by the second ligandpopulation.

The size of the substantially concentric deposition of the first ligandpopulation is to be selected to approximate the size of an immunologicalsynapse. In a human subject, the diameter of an immunological synapse ofa T cell is about 1-3 μm. While the deposition of the first ligandpopulation does not have to be circular, its approximate diameter canrange between about 10 nm to about 5 μm. In certain embodiments, theapproximate diameter of the deposition of the first ligand population isabout 0.5 μm to about 3.5 μm. In a particular embodiment, theapproximate diameter of the deposition of the first ligand population isabout 1.7 μm.

First and second ligand populations are generally comprised of one ormore ligands involved in T cell activation, one or more molecules towhich a ligand involved in T cell activation may be bound, or acombination thereof. Ligands involved in T cell activation include, butnot limited to anti-CD3 antibodies, anti-CD28 antibodies, anti-TCRantibodies, anti-CTLA4 antibody, and ligands comprising a generalintegrin-binding motif. Anti-CD3 antibodies include any antibody orother ligand capable of binding to a CD3 subunit (γ, δ, or ε) or CD3subunit dimer (ε/γ or ε/δ). Anti-CD28 antibodies include any antibody orother ligand capable of binding to CD28. Anti-TCR antibodies include anyantibody or other ligand capable of binding at least one T cell receptorsubunit (α and β). Anti-CTLA4 antibodies include any antibody or otherligand capable of binding to CTLA4 (CD152). Anti-CTLA4 antibodiesfurther include any antibody or other ligand capable of blocking CTLA4(CD152) signaling. Ligands comprising a general integrin-binding motifinclude, but are not limited to fibronectin, collagen, laminin,vitronectin, fibrinogen, and thrombospondin. Molecules to which a ligandinvolved in T cell activation, include, for example, biotin. An antibodyor other ligand described herein can be conjugated with streptavidin andincubated with a biomimetic Janus particle comprising biotin. Thestreptavidin-conjugated antibody or other ligand with then be bound tobiotin via biotin's strong interaction with streptavidin. Alternatively,a biomimetic Janus particle comprising biotin can be incubated withstreptavidin, followed by incubation with a biotinylated antibody orother ligand.

The first and second ligand populations can comprise of one or moreligands. Generally, the first and second ligand populations will differin their composition by at least one ligand. In certain embodiments, thefirst and second ligand populations will not share any common ligands.In yet other embodiments, the first and second ligand populationscomprise of a single type of ligand. In certain aspects, only one ofeither the first ligand population or second ligand population willcomprise an integrin-binding ligand. In a particular aspect, one ofeither the first ligand population or second ligand population comprisesthe integrin-binding ligand fibronectin.

As described above, the biomimetic Janus particle's first and secondligands are arranged either in a natural or reverse bull's eyeconfiguration, simulating either the natural or reverse organization ofligands in an immunological synapse of a natural antigen presentingcell. In an embodiment employing the natural bull's eye configuration,the first ligand population comprises at least one ligand capable ofbinding to at least one component of a T cell TCR complex, such as, forexample, an anti-CD3 antibody, an anti-CD28 antibody, an anti-TCRantibody, or an anti-CTLA4 antibody. In the natural bull's eyeconfiguration, the first ligand population can alternatively compriseone or more molecules to which a ligand capable of binding to at leastone component of a T cell TCR complex may be bound. For example, thefirst ligand population can comprise biotin. A streptavidin-conjugatedligand capable of binding to at least one component of a T cell TCRcomplex can then be bound to biotin. In the natural bull's eyeconfiguration, the second ligand population comprises at least oneligand comprising a general integrin-binding motif. In a particularembodiment, the first ligand population comprises an anti-CD3 antibodyand the second ligand population comprises fibronectin. In anotherparticular embodiment, the first ligand population comprises biotin, andthe second ligand population comprises fibronectin. An anti-CD3 antibodycan bound to biotin through a biotin-streptavidin interaction.

In an embodiment employing the reverse bull's eye configuration, thefirst ligand population comprises at least one ligand comprising ageneral integrin-binding motif, while the second ligand populationcomprises at least one ligand capable of binding to at least onecomponent of a T cell TCR complex. In the reverse bull's eyeconfiguration, the second ligand population can alternatively compriseone or more molecules to which a ligand capable of binding to at leastone component of a T cell TCR complex may be bound. For example, thesecond ligand population can comprise biotin. A streptavidin-conjugatedligand capable of binding to at least one component of a T cell TCRcomplex can then be bound to biotin. In a particular embodiment, thefirst ligand population comprises fibronectin, and the second ligandpopulation comprises an anti-CD3 antibody. In another particularembodiment, the first ligand population comprises fibronectin, and thesecond ligand population comprises biotin. An anti-CD3 antibody canbound to biotin through a biotin-streptavidin interaction.

The two types of patterned particles differentially direct the signalingproteins of T cells. When exposed to the reverse bull's eyeconfiguration, T cells exhibited annular or diffusive accumulation ofactin whereas the native bull's eye configuration exhibited either focalor diffusive accumulation of actin. Control T cells stimulated withparticles having uniform presentation of anti-CD3 and fibronectindisplayed only diffusive accumulation of PKC-θ. Similar results wereobserved with actin.

The data show that patterned particles modulate T cell activation fromthe outside in, so that the reverse bull's eye pattern, by preventing Tcell receptors and other signaling proteins from moving to the center ofthe immunological synapse, activates T cells more robustly than thenative pattern. Therefore, in a particular embodiment, T cells areactivated by biomimetic Janus particles patterned with a reverse bull'seye pattern. In another particular embodiment, biomimetic Janusparticles patterned with the native bull's eye pattern are used tosuppress an immune response.

The first and second ligand populations are patterned on nano- ormicro-sized particles. The nano- or micro-sized particles can be anyparticle capable of binding one or more ligands. Nano- or micro-sizedparticles useful as a base for biomimetic Janus particles describedherein include, but are not limited to, silica particles, polystyreneparticles, melamine resin particles, and polymethacrylate particles. Incertain embodiments, the nano- or micro-sized particles are silicaparticles.

Particles use as a base for biomimetic Janus particles described hereingenerally have a diameter of about 0.1 μm to about 20 μm. Particles canbe larger or smaller than this, but any such particle will not resemblethe size of a natural APC. In certain embodiments, particles havediameters of about 0.5 μm to about 5 μm. In one particular aspect,particles have a diameter of about 3 μm.

The biomimetic Janus particles described herein may be synthesized bymany different methods. These methods include, but are not limited to,microcontact printing, nanolithography, microlithography, and co-polymerself-assembly. Biomimetic Janus particles having either a native orreverse bull's eye ligand pattern may be synthesized by any one of thesemethods, or a combination thereof. The method of nano- ormicrolithography can be any such method known in the art, such asphotolithography, electron beam lithography, nanoimprint lithography,interference lithography, X-ray lithography, extreme ultravioletlithography, magnetolithography, and dip-pen lithography. Methods ofself-directed co-polymer assembly utilizing block co-polymers can alsobe used to synthesize biomimetic Janus particles. Any of these methodscan be used, where the method is capable of patterning one or moresubstantially concentric depositions of a first ligand population on anano- or micro-sized particle. In certain embodiments, eachsubstantially concentric deposition of the first ligand population has adiameter of about 10 nm to about 5 μm. In certain embodiments, theapproximate diameter of the deposition of the first ligand population isabout 0.5 μm to about 3.5 μm. In a particular embodiment, theapproximate diameter of the deposition of the first ligand population isabout 1.7 μm. In particular aspects, biomimetic Janus particles havingeither a native or reverse bull's eye pattern are synthesized bymicrocontact printing.

Generally, a biomimetic Janus particle is synthesized by forming atleast one substantially concentric pattern of a first ligand populationon an appropriate nano- or microparticle. After forming thesubstantially concentric pattern(s) of the first ligand population onthe particle, the particle is then incubated with a second ligandpopulation, so as to surround the substantially concentric pattern(s).It is preferable, although not required, that the second ligandpopulation not substantially overlap the substantially concentricpattern(s) of the first ligand population.

Characteristics of particles and ligand populations useful in thesynthesis of biomimetic are, unless otherwise noted, the same as thosedescribed above for a biomimetic Janus particle. For example, silica,polystyrene, melamine resin, or polymethacrylate particles having adiameter of about 0.1 μm to about 20 μm can be used in the methodsdescribed herein. First and second ligand populations useful for thesynthesis of a biomimetic Janus particle are the same as those describedabove.

Optionally, two or more substantially concentric patterns of the firstligand population are formed on a particle. The two or moresubstantially concentric patterns of the first ligand population aresurrounded by the second ligand population. In certain embodiments, eachsubstantially concentric deposition of ligand population comprises asingle type of ligand. Having multiple substantially concentric patternsof the first ligand population deposited on the particle has the benefitof increasing the probability of a T cell interacting with the native orreverse immunological synapse-mimicking bull's eye pattern formed on theparticle.

Where biomimetic Janus particles are patterned via microcontactprinting, a monolayer of particles is first deposited on a substrate,such as glass. As discussed and described above, particles of varioussizes and materials can be used. Where a monolayer of particles is to beformed on a substrate, as in microcontact printing, it is beneficial tosupply particles of substantially the same diameter. This can help toensure the deposition of a uniform monolayer on the substrate and tohelp ensure consistent microcontact printing. Particles can be cleanedprior to the formation of the monolayer on a substrate. Many differentsolvents can be used to clean particles, including water. Preferably,the solvent used to clean particles removes organic residues from theparticles. In some embodiments, the solvent is a strong oxidizing agentcapable of both removing organic residues and hydroxylating the surfaceof the particles, making them hydrophilic. In one embodiment, thesolvent used to clean the particles is piranha solution comprisingH₂SO₄/H₂O₂.

The substrate can be cleaned prior to the formation of the monolayer ofparticles thereon. Similarly to the particles, many different solventscan be used to clean the substrate. Preferably, a solvent used to cleanthe substrate removes organic residues. In some embodiments, the solventis a strong oxidizing agent capable of both removing organic residuesand hydroxylating the surface of the substrate, making it hydrophilic.In one embodiment, the solvent used to clean the substrate is piranhasolution comprising H₂SO₄/H₂O₂. Optionally, the substrate is cleanedwhile being maintained at an elevated temperature of about 30 to 100° C.In one aspect, the substrate is cleaned with piranha solution at atemperature of about 75° C. The substrate is cleaned for a period oftime sufficient to remove organic residues and hydroxylate the surfaceof the substrate. The period of time sufficient for cleaning will dependon the solvent used, and can be adjusted to ensure adequate cleaning.For example, where the cleaning solvent is piranha solution, thesubstrate is cleaned for at least 5 minutes. In one aspect where thesubstrate is cleaned with piranha solution, the substrate is cleaned forabout 15 minutes.

Following any substrate cleaning step, the substrate is rinsed in orderto remove the solvent from the substrate. The cleaned substrate can becleaned, for example, using distilled or ultrapure water.

Monolayers of particles may be formed on a substrate by any means knownin the art, including but not limited to, solvent evaporation and dipcoating.

A form of soft lithography, microcontact printing utilizes a polymerstamp coated, or “inked,” with substance, to form patterns on thesurface of a substrate. In its use to synthesize biomimetic Janusparticles, a polymer stamp inked with a first ligand population iscontacted with the monolayer of particles, which acts as themicrocontact printing “substrate.” Due to particles being spherical,this contacting step results in a substantially concentric deposition ofthe first ligand population being made on each particle of the monolayercontacted with the inked polymer stamp. Polymers useful for themicrocontact printing of biomimetic Janus particles include, but are notlimited to, silicon-based organic polymers and hydro-gel-formingpolymers. These polymers may be toughened or hardened (“cured”) bycross-linking polymer chains. In particular aspects,polydimethylsiloxane (PDMS) is used as the polymer stamp.

A cured polymer stamp is generally cut from a larger piece of curedpolymer, although cured polymer stamps of a particular size can beformed directly. In some embodiments, the cured polymer stamp has asurface area of about 0.5 to about 10 cm². In one aspect, the section ofcured polymer has a surface area of about 1 cm². In one particularembodiment, the section of cured polymer comprises PDMS having a surfacearea of about 1 cm².

A section of cured polymer can be treated in order to make the surfaceof the section of cured polymer hydrophilic, which facilitates thecoating or “inking” step, allowing the first ligand population to moreeasily adsorb to the surface of the cured polymer stamp. Treatment ofthe cured section can included treatment with a strong oxidizing agentcapable of hydroxylating the surface of the section of cured polymer,thereby making the treated surface of the section of cured polymerhydrophilic. In a particular embodiment, a cured section of polymer ishydroxylated by treating it with piranha solution comprising H₂SO₄/H₂O₂.The strength of the oxidizing agent can be adjusted to achieve a desiredresult. For example, in one embodiment, the piranha solution comprises a3:1 solution of H₂SO₄/H₂O₂.

Before pressing the section of cured polymer onto the monolayer ofparticles, the section of cured polymer is first “inked,” or coated,with a ligand. Contacting the section of inked, cured polymer with theparticles thereafter causes the individual particles of the monolayer ofparticles to partially embed in the inked, cured polymer. Removing theparticle from the inked, cured polymer leaves a substantially concentricdeposit of ligand on the particle.

The section of cured polymer is inked with a first ligand population asdescribed above. Briefly, the first ligand population comprises one ormore ligands such as an anti-CD3 antibody, an anti-CD28 antibody, ananti-TCR antibody, an anti-CTLA4 antibody, a ligand comprising a generalintegrin-binding motif, or biotin. The section can also be inked withany other ligand not listed that is known to be involved in T cellactivation at the immunological synapse. The section of cured polymermay be inked with the ligand by incubating the section of cured polymer,or a surface thereof, in the ligand. Where the particles are to bepatterned with a natural bull's eye pattern, the first ligand populationcomprises a ligand capable of binding to at least one component of a Tcell TCR complex, such as an anti-CD3 antibody, or biotin. As describedabove, through a biotin-streptavidin interaction, a ligand capable ofbinding to at least one component of a T cell TCR complex can then bebound to biotin. In other embodiments, where the particles are to bepatterned with a reverse bull's eye pattern, the section of curedpolymer is inked with a first ligand population comprising at least oneligand having a general integrin-binding motif.

Prior to contacting the inked section of cured polymer with themonolayer of particles, the inked section of cured polymer may be dried.Drying the inked, cured section of polymer results in a more precise andcontrollable transfer of the ligand from the inked section of curedpolymer to the particles. An inked, cured section of polymer can bedried, for example, under a stream of nitrogen prior to pressing thesection onto a monolayer of particles.

To transfer the ligand population bound (inked) to the section of curedpolymer, the inked section of cured polymer is contacted with themonolayer of particles. This is done under a known pressure, whichallows for the control of the diameter of the substantially concentricarea of ligand deposited on a particle. An increase in pressure willresult in particles being further embedded in the section of curedpolymer, resulting in deposition of a substantially concentric patternhaving a larger diameter. The diameter of the substantially concentricarea of ligand deposited on a particle can also be controlled bycontrolling the stiffness of the cured polymer, where under the sameapplied pressure, a softer polymer will result in a substantiallyconcentric pattern having a larger diameter. Methods of controlling thestiffness of a cured polymer are known in the art. For example, theratio of monomer to crosslinker can be adjusted to provide a polymer ofdesired stiffness.

In certain embodiments, the cured section of polymer incubated with thefirst ligand is pressed against the monolayer of particles at a pressureof about 0.5×10⁴ to 2.5×10⁴ Pa. In a particular embodiment, pressing asection of inked, cured PDMS prepared as described in the Examples,against a monolayer of particles at a pressure of 1.5×10⁴ results in thedeposition of a substantially concentric pattern of about 1.7 μm on theparticle, which is a similar size to the central protein domain found inthe immunological synapse. Utilizing a lesser pressure will generallyresult in a substantially concentric area of ligand having a smallerdiameter, as will using a stiffer section of polymer. Utilizing a higherpressure, or a softer polymer, will generally result in a substantiallyconcentric area of ligand deposited on the particle to have a largerdiameter.

The pressure applied to the inked, cured section of polymer, thestiffness of the section of polymer, or a combination thereof, can beadjusted to provide a substantially concentric deposition of the firstligand population on a particle having a diameter of about 10 nm toabout 5 μm. In certain aspects, the substantially concentric depositionof the first ligand population on the particle is about 0.5 to about 3.5μm. In a particular embodiment, the substantially concentric depositionof the ligand on a particle mimics the diameter of an immunologicalsynapse of a T cell of a subject. In a human subject, the diameter of animmunological synapse of a T cell is about 1-3 μm. Therefore, in aparticular embodiment, a section of cured PDMS inked with a ligand isused to generate a substantially concentric deposition of ligand on aparticle, wherein the substantially concentric deposition of ligand onthe particle has a diameter of about 1 to about 3 μm.

Following contacting the inked, cured section of polymer with themonolayer of particles, the monolayer of particles is removed from thesubstrate, and the particles are incubated in a second ligand. Theparticles can either remain embedded in the inked, cured section ofpolymer during incubation with the second ligand population, or can bereleased from the inked cured section of polymer prior to incubationwith the second ligand population. In either instance, the second ligandwill adsorb to the particle, surrounding the substantially concentricpattern of first ligand deposited on the particles by the inked, curedsection of polymer. Where particles remain embedded in the inked, curedsection of polymer, following incubation with the second ligandpopulation, the particles are released from the inked, cured section ofpolymer. Embedded particles can be released by, for example, sonication.Optionally, where particles remain embedded in the inked, cured sectionof polymer during incubation with the second ligand population, thereleased particles are re-incubated with the second ligand population.This second incubation with the second ligand population minimizes anygaps between the first ligand population and the second ligandpopulation that can occur when the particles are incubated with thesecond ligand population while still embedded in the inked, curedsection of polymer.

Where the particles are to be patterned with a natural bull's eyepattern, the second ligand population comprises at least one ligandhaving a general integrin-binding motif. In other embodiments, where theparticles are to be patterned with a reverse bull's eye pattern, thesecond ligand population comprises at least one ligand capable ofbinding to at least one component of a T cell TCR complex, such as ananti-CD3 antibody, or biotin. As described above, through abiotin-streptavidin interaction, a ligand capable of binding to at leastone component of a T cell TCR complex can then be bound to biotin.

Incubation with the second ligand population is for a period of time toallow for the second ligand population to adsorb to particle. In certainembodiments, incubation with the second ligand population is for about0.5 to about 3 hours. In a particular embodiment, incubation with thesecond ligand population is for about 1.5 hours.

Optionally, after particles have been patterned with the first andsecond ligands, and, if embedded in the inked, cured section of polymer,have been released from the same, the patterned particles can undergopassivation, for example, by treating the particles with PBS buffercomprising bovine serum albumin.

Optionally, the process of contacting an inked, cured section of polymerwith a particle is repeated one or more times, releasing the particlefrom the inked, cured section of polymer between each repetition.Following contacting a particle two or more times with an inked, curedsection of polymer, the particle is incubated with the second ligandpopulation. This process results in two or more substantially concentricdepositions of first ligand population surrounded by a second ligandpopulation (FIG. 1F). In another embodiment, a different first ligand isinked on a cured polymer for use in the repeated contacting step,wherein each substantially concentric deposition of ligand populationcomprises a single type of ligand. Having multiple substantiallyconcentric patterns of the first ligand population deposited on theparticle has the benefit of increasing the probability of a T cellinteracting with the native or reverse immunological synapse-mimickingbull's eye pattern formed on the particle.

In embodiments where either the first or second ligand populationcomprises biotin, particles to which biotin are bound are furtherincubated with either a streptavidin-conjugated ligand or simply withstreptavidin. When simply incubated with streptavidin, a furtherincubation step with a biotinylated ligand is required. These stepsresult in binding one or more ligands involved in T cell activation tothe particle via biotin.

In yet other embodiments, co-polymer self-assembly is employed topattern at least one substantially concentric pattern of a first ligandpopulation on a particle. In one embodiment, a first ligand population,as described above, is bound to a first block copolymer. The first blockcopolymer is combined with a second block copolymer to form a sphericalbiomimetic Janus particle having at least one substantially concentricpattern of the first ligand population.

The resulting spherical biomimetic Janus particle having at least onesubstantially concentric pattern of the first ligand population can thenbe incubated with a second ligand population, as described above.Alternatively, a second ligand population, can be bound to a secondblock copolymer prior to being combined with the first block copolymer.Combining the first block copolymer, to which is bound a first ligandpopulation, with the second block copolymer, to which is bound a secondligand population, results in a spherical biomimetic Janus particleshave at least one substantially concentric pattern of the first ligandpopulation surrounded by the second ligand population.

In certain embodiments, the first and second block copolymers undergodirected self-assembly.

Also contemplated herein are biomimetic Janus particles produced by anyof the methods described herein. Other aspects contemplated hereininclude a compound comprising one or more biomimetic Janus particlesdescribed herein and/or one or more biomimetic Janus particles producedby any of the methods described herein. Compounds comprising biomimeticJanus particles can be formulated for various applications such as, forexample, in vitro activation of T cells.

Biomimetic Janus particles described herein, whether synthesized by amethod described herein or not, are useful for activating a populationof T cells in vitro. Efforts to amplify T cell response to diseasesincluding cancer, autoimmune disease, and viral infection often focus onimmunotherapies involving transferring activated T cells into a subject.Signals that T cells receive from native natural antigen presenting cell(APC) during and after their initial encounter with an antigen caninfluence their programming and subsequent therapeutic efficiency. Theinability to regulate the signals and interactions provided by naturallyoccurring APCs has resulted in increased interest in the use ofartificial APCs to allow for greater control over T cell signaling andfacilitate the generation of optimally effective T cells forimmunotherapy. In addition, isolation of native APCs for T cell-basedimmunotherapies is time-consuming, expensive, and difficult toreproduce.

It is known that T cell activation involves the spatial organization ofligands. Initial ligand-receptor recognition leads to the formation of amicron-sized membrane junction known as the immunological synapse. As Tcell signaling proceeds, ligand-bound membrane receptors and signalingproteins, such as TCRs, co-stimulatory receptors and adhesion molecules,are clustered and reorganized in the synapse to form a “bull's eye”pattern of several distinct concentric domains (FIG. 1A). Ligand-boundTCRs are transported from cell periphery to accumulate in the centraldomain of the immunological synapse, while integrins and manycytoskeletal proteins are enriched in a ring structure surrounding theTCRs. The protein spatial organization is a reflection of long-rangemolecular interactions that take place in T cell activation.Additionally, the immunological synapse formation, by controlling whichproteins come together and when they are apart, is a regulatorycheckpoint for T cell stimulation.

T cells and other immune cells exhibit different levels of activationwhen they encountered micropatterned “islands” of protein ligandsdesigned to separate or co-localize different membrane receptor on flatsurfaces. Cell signaling is prolonged on micropatterned lipid bilayerson which transport of receptor-ligand pairs was constrained by metalgrids. Ligand density and spacing, controlled by using nanoparticlearrays, also affect T cell activation. Several studies involvingtwo-dimensional surfaces have demonstrated the potential of modulating Tcell activation by spatially manipulating the immunological synapseformation. However, these two-dimensional arrangements do not mimic thethree-dimensional structure of native APCs

The biomimetic Janus particles described herein either mimic the ligandorganization found in a native immunological synapses, or are arrangedin reverse relative to a native immunological synapse. The biomimeticJanus particles described herein are useful for activating T cells invitro. T cells, once activated in vitro by a biomimetic Janus particlesdescribed herein, may be used in an immunotherapy in a subject in needthereof.

T cells can be activated by one or more biomimetic Janus particlesdescribed herein. In particular, the biomimetic Janus particlesdescribed herein are useful for activating T cells in vitro. T cellsactivated in vitro by biomimetic Janus particles described herein can beused in an immunotherapy administered to a subject in need thereof.

T cells derived from several sources can be cultured, activated bybiomimetic Janus particles described herein, and utilized in animmunotherapy. T cells to be activated can be autologous T cellsisolated from a subject in need of an immunotherapy, heterologous Tcells derived from a source other than the subject in need of animmunotherapy, or a combination thereof. Naïve T cells, CD8⁺ T Cells,CD4⁺ T cells, or any combination thereof, can also be activated bybiomimetic Janus particles described herein.

In certain embodiments, the T cells to be activated are antigen-specificT cells. Antigen-specific T cells can be generated by methods known inthe art, including but not limited to, identifying and cloning T cellsfrom subjects with particularly good antigen responses, generatingchimeric antigen receptors and transfecting cultured T cells with thechimeric antigen receptor specific for a particular antigen of interest,and isolating T cell receptors from humanized mice that have been primedto recognize a particular antigen of interest, wherein the mice expresshuman MHC class I or MHC class II molecules and can be immunized withthe particular antigen of interest, and wherein mouse T cells specificfor the MHC-restricted epitope of interest can then be isolated, andtheir TCR genes cloned into recombinant vectors that can be used togenetically engineer cultured T cells. In one preferred embodiment, theT cells are tumor-antigen specific T cells.

To activate T cells using biomimetic Janus particles described herein,the biomimetic Janus particles are added to cultured T cells. Thecultured T cells are incubated with the biomimetic Janus particles forabout 5 minutes to about 2 weeks to allow activation. The biomimeticJanus particles interact with the cultured T cell, mimicking theimmunological synapse formation between a T cell and a natural antigenpresenting cell. The biomimetic Janus particles direct T cell proteinmovement and signaling to activate the T cell. In certain embodiments,cultured T cells are incubated with the biomimetic Janus particlesdescribed herein for about 1 to about 60 hours, or for about 24 to about48 hours.

The population of cultured T cells can be rinsed following incubationwith the biomimetic Janus particles described herein to remove theparticles from the population of T cells. Rinsing is preferable when theT cells are to be introduced into a subject as part of an immunotherapy.

T cells activated by the biomimetic Janus particles described herein canbe used in immunotherapies including, but not limited to adoptiveimmunotherapy for cancer, tolerance induction in autoimmune disease,autologous immune enhancement therapy, and viral infectionimmunotherapy. Such immunotherapies are known in the art (see, e.g.,Restifo et al., (2012) Nat Rev Immunol, March 22; 12(4):269-81; Maus etal., (2014) Annu Rev Immunol, 32:189-225; Singer et al., (2014) FrontImmun, 5:46). The biomimetic Janus particles described herein can beused or adopted for use in such therapies. Optionally, prior toadministering an immunotherapy comprising administering activated Tcells described herein to a patient, the patient is subjected to a stepof preparative lymphodepletion (Gattinoni et al., (2006) Nat RevImmunol, May; 6(5):383-93). Modifications to the biomimetic Janusparticles can be made to tailor their use to a specific situation. Suchmodifications are contemplated herein, and do not depart from theessential scope of the present disclosure.

EXAMPLES

The materials, methods, and embodiments described herein are furtherdefined in the following Examples. Certain embodiments of the presentinvention are defined in the Examples herein. It should be understoodthat these Examples, while indicating certain embodiments of theinvention, are given by way of illustration only. From the discussionherein and these Examples, one skilled in the art can ascertain theessential characteristics of the present invention and without departingfrom the spirit and scope thereof, can make various changes andmodifications of the invention to adapt it to various usages andconditions, such as for the use in activating T cells for the variousimmunotherapies described above.

Example 1—Materials and Methods

Reagents and Cells.

SPHERO™ silica particles (5% w/v) were purchased from Spheroptech Inc.(Lake Forest, Ill.). Bovine serum albumin (BSA) and BSA-biotin werepurchased from Thermo Scientific (Waltham, Mass.). BiotinN-hydroxysuccinimide ester (biotin-NHS) was purchased from Sigma-Aldrich(St. Louis, Mo.). Anti-human CD3 (anti-CD3) OKT antibody was purchasedfrom eBioscience (San Diego, Calif.) and conjugated with biotin-NHS.Phalloidin Alexa 647 conjugate was purchased from Cell SignalingTechnology (Danvers, Mass.). Fibronectin (human) was obtained from BDBiosciences (San Jose, Calif.) and further conjugated with Alexa Fluor®488 carboxylic acid, succinimidyl ester. Protein kinase C (PKC)-θantibody (C-19) was purchased from Santa Cruz Biotech (Dallas, Tex.).Fluo-4 AM and Alexa Fluor® 647 Chicken Anti-Goat IgG (H+L) werepurchased from Invitrogen (Eugene, Oreg.). Polydimethylsiloxane (PDMS;Sylgard 184) was obtained from Dow Corning (Midland, Mich.) and used at2:1 (w:w) base-to-curing-agent ratio.

Jurkat T cells (clone E6-1) were originally purchased from ATCC(Manassas, Va.). Jurkat T cells were cultured in RPMI 1640 completegrowth media supplemented with 10% fetal bovine serum (FBS), 1 mM sodiumpyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin. Ultrapurewater (resistivity of 18.2 MΩ·cm) was used. Calcium-containing imagingbuffer (121 mM NaCl, 6 mM NaHCO₃, 5.4 mM KCl, 5.5 mM D-glucose, 0.8 mMMgCl₂, 25 mM HEPES, 1.8 mM CaCl₂, pH 7.4) was used for live cell imagingexperiments. Live cell imaging chambers and temperature controllers werepurchased from Bioptechs (Butler, Pa.).

Microcontact Printing of Janus Particles.

Glass microscope slides were treated with piranha solution of H₂SO₄/H₂O₂(30%), 3:1 at 75° C. for 15 minutes and rinsed in ultrapure water.Silica particles of 3-μm in diameter were cleaned with piranha solutionand deposited via solvent evaporation on a clean glass microscope slideto form a monolayer. Sylgard 184 base and curing agent were mixed at 2:1(w:w) ratio in a plastic cup, poured into a flat petridish, degassed invacuum until no bubbles were visible, and cured at 65° C. for 12 hours.Small sections of the PDMS stamp (1 cm×1 cm) were cut out and treatedwith piranha solution to make the surface hydrophilic.

To generate the reverse “bull's eye” pattern in which a fibronectinpatch is surrounded by anti-CD3 molecules, the top surface of a PDMSstamp was incubated with Alexa Fluor® 488-fibronectin solution (2 μg/mL)for at least 20 minutes, dried under a stream of nitrogen, andimmediately pressed against the monolayer of silica particles at apressure of 1.5×10⁴ Pa. After 3 minutes, the stamp with embedded silicaparticles was peeled off from the substrate and incubated with AlexaFluor® 568-BSA-biotin solution (16.5 μg/mL) for 1.5 hours. Particleswere sonicated off the PDMS stamp and harvested in 1×PBS buffercontaining BSA (0.005%, w/v) for passivation.

Janus particles with the native “bull's eye” pattern were preparedthrough the same procedure using PDMS stamps inked with BSA-biotin. Manyparticles exhibited a gap between the BSA-biotin patch andfibronectin-covered surface after incubation with fibronectin becausefibronectin molecules were prevented from adsorbing onto the particlesurface near the PDMS stamp due to the large size. The gap was filled byadditional incubation in a diluted fibronectin solution (1 μg/mL in1×PBS buffer) for 2 hours. The Janus particles were then furtherfunctionalized with streptavidin (100 nM) and biotinylated anti-CD3 (20nM).

Calcium Imaging and Analysis.

Jurkat T cells were serum starved in serum-free cell media at 37° C. for2 hours before imaging. To load cells with the intracellular Ca²⁺indicator Fluo-4 AM, 1 million cells were incubated with 5 μg/mL Fluo-4in serum-free cell media at 37° C. for 30 minutes, washed, and thenincubated in serum-rich cell media at 37° C. for another 30 minutes toallow complete de-esterification of intracellular AM esters. The Fluo-4loaded T cells were suspended in 1× imaging buffer and added into animaging chamber at 37° C. after the addition of Janus particles.

Concentrations of Janus particles and cells were kept the same in allexperiments. Time-lapse multi-channel epifluorescence microscopy imageswere immediately taken with a Nikon Eclipse Ti microscope system that isequipped with an iXon3 EMCCD Camera (Andor Technology) and a Nikon PlanAPO 40×/0.95 N.A objective or a Nikon Plan Apo 100×/1.49 N.A TIRFobjective. Images were acquired with 100 ms exposure time and 2 sinterval time. Imaging chambers were maintained at 37° C. with a heater.

A Matlab script was used to quantify the fluorescence intensity ofindividual cells as a function of time. The algorithm detected theoutline of individual cells and calculated the average fluorescenceintensity per pixel for each cell. Cells that were not in contact withany particles were removed in image processing. Due to uneven loading ofdyes into cells, some cells appeared brighter than others. A basalintensity was obtained by averaging the fluorescence intensity of thefirst 25 imaging frames before the first calcium peaks. The averagefluorescence intensity of each cell was then normalized by the basalintensity to enable comparison of the calcium signaling between cellsand samples.

Immunofluorescence Staining and Confocal Fluorescence Imaging.

Jurkat T cells were serum starved at 37° C. for 2 hours before mixingwith particles. 5 million cells were mixed with Janus particles in 1×imaging buffer solution for 4 minutes before fixation. Cells were fixedwith 2% (w/v) paraformaldehyde (PFA) on ice for 15 minutes,permeabilized with 0.01% Triton X-100 for a few seconds, and blockedwith 1% bovine serum albumin (BSA) for 1 hour. Actin was stained byincubation with 0.32 μg/mL phalloidin-Alexa Fluor® 647 for 30 minutes atroom temperature. To label PKC-θ, permeabilized cells were incubatedwith 1 μg/mL PKC-θ antibody at room temperature for 2 hours, washed with1×PBS solution for 3 times, blocked with 1% BSA for 30 minutes, andincubated with 1 μg/mL Alexa Fluor® 647 Chicken Anti-Goat IgG (H+L) atroom temperature for 1 hour.

Confocal scanning fluorescence imaging was done on a Nikon A1R-A1confocal microscope system equipped with a Nikon 100× oil-immersedobjective and a Hamamatsu C11440 camera (Light Microscopy ImagingCenter, Indiana University). Alternative scanning mode was used to avoidpossible crosstalk between the Alexa 568 and Alexa 647 channels. Z-scanstacks were acquired with a 0.15 μm z-axis increment. Images wereanalyzed with ImageJ software.

Example II—Design and Fabrication of Biomimetic Janus Particles

T cell receptors (TCRs) and integrins are two membrane receptors thatare known to play important roles in T cell activation and inimmunological synapse formation. Anti-CD3 antibodies and fibronectinmolecules were used in this study as ligands for TCRs and integrins,respectively. Anti-CD3 crosslinks CD3 subunits in the TCR complex andtriggers T cell activation. Fibronectin is an adhesion molecule thatbinds α4β1 and α5β1 integrins on the Jurkat T cell membrane.

Two types of “bull's eye” protein patterns were generated onmicroparticles. One pattern resembles the native immunological synapse,in which anti-CD3 is enriched in the central domain and fibronectinaccumulates in the surrounding region. The other protein pattern is thereverse type, in which a patch of fibronectin is surrounded by a fieldof anti-CD3.

A microcontact printing method (FIG. 1C) was developed to fabricate the“bull's eye” Janus particles, such that the protein patches were ofsimilar size to the protein domains of a native immunological synapse.Firstly, a monolayer of 3-μm silica particles was deposited on a flatsubstrate. To generate the native “bull's eye” pattern, a BSA-biotinpatch was generated by bringing a polydimethylsiloxane (PDMS) stamp thatwas “inked” with BSA-biotin solution into brief contact with theparticles. The PDMS stamp was then lifted, removing the monolayer fromthe substrate, leaving partially embedded particles in the stamp. Theexposed surfaces of the partially embedded particles were subsequentlycoated with fibronectin by incubation.

Janus particles with the reverse “bull's eye” pattern were fabricatedusing the same general procedure, during which the fibronectin patcheswere printed with a PDMS stamp, followed by BSA-biotin adsorption ontothe exposed surface of particles. For both native and reverse bull's eyepatterns, biotinylated anti-CD3 was conjugated with BSA-biotin viastreptavidin linkers.

The size of protein patches can be adjusted by altering the stiffness ofPDMS stamps, with softer stamps generating larger patches. In aparticular experiment, a relatively hard stamp made of 2:1monomer-to-crosslinker ratio resulted in protein patches having averagediameters of (1.7±0.3) μm (FIG. 4), a similar size to the centralprotein domain found in the native immunological synapse. The morphologyof the native and reverse “bull's eye” patterns was confirmed using 3-Dfluorescence confocal scanning microscopy (FIGS. 1D-1E). Those imagesalso show that cross-contamination of the two protein domains wasnegligible.

The spatial organization of proteins in T cells was determined tofollows the patterns of anti-CD3 and fibronectin on the Janus particlesurface. The clustering of two intracellular signaling proteins, actinand protein kinase C (PKC)-θ, was examined. Neither of the proteins aremembrane bound, but are known to be associated with TCRs and spatiallysegregate in the immunological synapse. Specifically, actin is acytoskeletal network that supports clustering and translocation ofmembrane receptors including TCRs. Actin distributes across the entireimmunological synapse at the beginning of T cell activation, butaccumulates in a ring structure around the central accumulation of TCRsin the mature synapse. PKC-θ is a signaling protein required for T cellactivation and survival; its intracellular clustering follows TCRs atinitial T cell activation.

Example III—Activation of T Cells by Biomimetic Janus Particles

In order to capture the early TCR activation stage and to preventcomplete engulfment of particles, Jurkat T cells were fixed at 4 minutesafter mixing them with particles. Cells that faced the “bull's eye”patterns were observed for data collection, although particlesinteracted with cells at random orientations. Morphologies of actin andPKC-θ were grouped into three categories: diffusive, focal, and annular.In a majority of the cells (80% of 51 cells) that were in contact withthe native “bull's eye” pattern, actin appeared diffusive over theentire contact area (FIG. 2A). Other cells (20% of 51 cells) exhibitedfocal accumulation of actin, which colocalized with the anti-CD3 patchon the particle. However, cells that were in contact with the reverse“bull's eye” pattern only exhibited either annular (52% of 55 cells) ordiffusive (48% of 55 cells) morphology. Actin does not colocalize withfibronectin, but largely follows the anti-CD3 patterns on the particles.

Similar phenomena were observed for PKC-θ. Intracellular clustering ofPKC-θ was either focal (16% of 44 cells) or diffusive (84% of 44 cells)in cells that were activated by the native “bull's eye” pattern (FIG.2B). However, cells that were activated by the reverse “bull's eye”pattern exhibited only annular (51% of 39 cells) or diffusive (49% of 39cells) accumulation of PKC-θ. To confirm that the intracellularclustering of actin and PKC-θ was indeed altered by the ligand patternson particles, T cells were stimulated with particles that have uniformpresentation of anti-CD3 and fibronectin. Only diffusive morphology ofactin and PKC-θ was observed (FIGS. 5 and 6). It is evident that theintracellular clustering of the two key signaling proteins, actin andPKC-θ, is directed by the “bull's eye” protein patterns on particles.

The central accumulation of TCRs is known to lead to signalingtermination. Prolonged T cell signaling has been observed when the Tcell receptors were prevented from moving toward the center of theimmunological synapse. The “bull's eye” patterns on particles describedherein directed the clustering of two intracellular proteins: actin andPKC-θ.

T cell activation was quantified by measuring intracellular Ca²⁺elevation. It is known that T cell activation leads to rapid entry ofcalcium ions into cytosol from both the endoplasmic reticulum (ER) andthe extracellular solution. The amplitude and duration of the calciumelevation in T cells are directly related with the strength of T cellactivation, with stronger stimuli giving more prominent and sustainedcalcium influx. Intracellular calcium elevation in single T cells wasimaged by using the calcium-sensitive dye Fluo-4 acetoxymethyl ester(AM), whose fluorescence emission increases upon binding Ca²⁺. As shownin FIG. 3A, when a Jurkat T cell made the initial contact with thereverse “bull's eye” pattern on a particle, a rapid increase offluorescence intensity immediately followed, indicating a large influxof Ca²⁺ into the cytosol. As T cell signaling proceeded, Fluo-4fluorescence intensity gradually decreased. Plotting the fluorescenceintensity (normalized to baseline) as a function of time showed thetemporal changes of intracellular [Ca²⁺] during T cell activation.Calcium influx peaked within 2 minutes after the initial cell-particlecontact, sustained for 1-3 minutes and gradually returned to a basallevel. Such calcium influx is characteristic of normal T cellactivation. In contrast, T cells that were stimulated by the native“bull's eye” particles exhibited transient calcium peaks of lowerintensities (FIG. 3C). A delay between the initial cell-particle contactand the first calcium peak was also typical, indicative of a phaseduring which T cells searched for stimulatory signal from anti-CD3. Thetwo cell-particle pairs are each representative of over 30 cells thatwere in contact with the “bull's eye” pattern.

To further elucidate the global response of Ca²⁺ signaling upon T cellstimulation by the “bull's eye” Janus particles, a large population of Tcells was analyzed, and their normalized fluorescence intensitiesplotted as a function of time in heat maps (FIGS. 3B and 3D). Calciumelevation differed in T cells stimulated by the two types ofparticles—the reverse “bull's eye” Janus particles, in comparison to thenative type—lead to more intense calcium influx in a larger fraction ofcells. In control experiments in which particles were uniformly coatedwith anti-CD3, fibronectin, or BSA, it was confirmed that Jurkat T cellswere activated by anti-CD3 but not by fibronectin alone (FIG. 7). Thecell-to-cell variation of calcium signaling is characteristic of T cellactivation and in agreement with previous reports. Given that thecalcium heat maps were obtained from all particle-cell orientations, itis possible that the difference of calcium profiles is partiallyaugmented by the different surface coverage of anti-CD3 on the two typesof particles.

Both single-cell and bulk results confirmed that the spatialpresentation of anti-CD3 and fibronectin on the particles influenced thestrength and duration of calcium signaling during T cell activation. Thestronger T cell activation by the reverse “bull's eye” particles agreeswith the modulated annular localization of actin and PKC-θ, confirmingthat the “bull's eye” Janus particles modulate T cell activation bydictating the spatial organization of signaling proteins.

Using a microcontact printing method, micron-sized particles weregenerated as artificial antigen presenting cells that display “bull'seye” patterns of protein ligands on the particle surface. One patternmimics the native organization of proteins in the immunological synapse,while the other is a reverse pattern of the same protein's ligands. Itwas found that the reverse “bull's eye” Janus particles lead to moreintense and sustaining T cell calcium signaling than the native type.This is due to the differential T cell activation to the modulatedintracellular localization of signaling proteins, which was confirmedfor actin and PKC-θ. These results demonstrate that fixed arrangement ofprotein ligands on particle surfaces can be used to modulate T cellactivation from outside in. This shows how multi-functional Janusparticles can be designed as artificial antigen-presenting cells forfine-tuning T cell activation.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

1.-31. (canceled)
 32. A method for producing a biomimetic Janusparticle, the method comprising the steps of a) forming at least onesubstantially concentric pattern of a first ligand population on atleast one particle, where the first ligand population comprises one ormore ligands involved in T cell activation and/or one or more moleculesto which a ligand involved in T cell activation may be bound; and b)incubating the at least one particle following completion of step a)with a second ligand population, where the second ligand population isdifferent than the first ligand population and does not substantiallyoverlap the substantially concentric pattern of the first ligandpopulation, and Where the second ligand population comprises one or moreligand involved in T cell activation and/or one or more molecules towhich a ligand involved in T cell activation may be bound.
 33. Themethod of claim 1 where the at the least one substantially concentricpattern of the first ligand population is deposited by means selectedfrom the group consisting of microcontact printing, nanolithography,microlithography, photolithography, electron beam lithography,nanoimprint lithography, interference lithography, X-ray lithography,extreme ultraviolet lithography, magnetolithography, and dippenlithography.
 34. The method of claim 1 where the at least one particleis a microparticle or a nanoparticle.
 35. The method of claim 1 wherethe at least one particle is selected from the group consisting of asilica particle, a polymer-based particle, and a magnetic particle. 36.The method of claim 1 where the at least one particle is a magneticparticle.
 37. The method of claim 1 where the at least one particle hasa diameter within a range selected from the group consisting of about0.1 μm to about 20 μm, and about 0.5 to about 5 μm.
 38. The method ofclaim 1 where the at least one particle has a diameter of about 3 μm.39. The method of claim 1 where the at least one particle is a silicaparticle having a diameter of about 3 μm.
 40. The method of claim 1where the at least one particle is a magnetic particle having a diameterof about 3 μm.
 41. The method of claim 1 where the least onesubstantially concentric pattern of the first ligand population has adiameter within a range selected from the group consisting of about 10nm to about 5 μm, and about 0.5 μm to about 3.5 μm.
 42. The method ofclaim 1 where the at least one substantially concentric pattern of thefirst ligand population has a diameter of about 1.7 μm.
 43. The methodof claim 1 where the one or more ligands involved in T cell activationis selected from the group consisting of anti-CD3 antibody, anti-CD28antibody, anti-TCR antibody, and anti-CTLA4 antibody, and a ligandcomprising a general integrin-binding motif, and where the one or moremolecules to which a ligand involved in T cell activation may be boundis biotin.
 44. The method of claim 1 where the first ligand populationcomprises one or more ligands comprising a general integrin-bindingmotif and the second ligand population comprises one or more ligandscapable of binding to at least one component of a T cell TCR complex.45. The method of claim 1 where the first ligand population comprisesone or more ligands capable of binding to at least one component of a Tcell TCR complex and the second ligand population comprises one or moreligands comprising a general integrin-binding motif.
 46. The method ofclaim 43 where the ligand comprising a general integrin-binding motif isselected from the group consisting of fibronectin, collagen, laminin,vitronectin, fibrinogen, and thrombospondin.
 47. The method of claim 44where the first ligand population comprises fibronectin and the secondligand population comprises an anti-CD3 antibody.
 48. The method ofclaim 45 where the first ligand population comprises an anti-CD3antibody and the second ligand population comprises fibronectin.
 49. Themethod of claim 44 where the one or more ligands capable of binding toat least One component of a T cell TCR complex is selected from thegroup consisting of anti-CD3 antibody, anti-CD28 antibody, anti-TCRantibody, and anti-CTLA4 antibody.
 50. A biomimetic Janus particleproduced by claim
 1. 51. A composition comprising one or more Janusparticles produced by claim 1.